Pancreatic Stellate Cells Research Articles

Discovery of YS-1 as a cell line of gastric inflammatory cancer-associated fibroblasts.

Inflammatory cancer-associated fibroblasts (iCAFs) was first identified by co-culture of pancreatic stellate cells and tumor organoids. The key feature of iCAFs is IL-6high/αSMAlow. We examine this phenomenon in gastric cancer using two cell lines of gastric fibroblasts (HGF and YS-1). HGF or YS-1 were co-cultured with MKN7 (a gastric adenocarcinoma cell line) in Matrigel. IL-6 protein levels in the culture supernatant were measured by ELISA. The increased production of IL-6 was not observed in any of the combinations. Instead, the supernatant of YS-1 exhibited the higher levels of IL-6. YS-1 showed IL-6high/αSMA (ACTA2)low in real-time PCR, mRNA-seq and immunohistochemistry. In mRNA-seq, iCAFs-associated genes and signaling pathways were up-regulated in YS-1. No transition to myofibroblastic phenotype was observed by monolayer culture, or the exposure to sonic hedgehog (SHH) or TGF-β. YS-1 conditioned medium induced changes of morphology and stem-ness/differentiation in NUGC-3 (a human gastric adenocarcinoma cell line) and UBE6T-15 (a human bone marrow-derived mesenchymal stem cell line). YS-1 is a stable cell line of gastric iCAFs. This discovery will promote further research on iCAFs for many researchers.

New avenues for the treatment of immunotherapy-resistant pancreatic cancer.

Pancreatic cancer (PC) is characterized by its extremely aggressive nature and ranks 14th in the number of new cancer cases worldwide. However, due to its complexity, it ranks 7th in the list of the most lethal cancers worldwide. The pathogenesis of PC involves several complex processes, including familial genetic factors associated with risk factors such as obesity, diabetes mellitus, chronic pancreatitis, and smoking. Mutations in genes such as KRAS, TP53, and SMAD4 are linked to the appearance of malignant cells that generate pancreatic lesions and, consequently, cancer. In this context, some therapies are used for PC, one of which is immunotherapy, which is extremely promising in various other types of cancer but has shown little response in the treatment of PC due to various resistance mechanisms that contribute to a drop in immunotherapy efficiency. It is therefore clear that the tumor microenvironment (TME) has a huge impact on the resistance process, since cellular and non-cellular elements create an immunosuppressive environment, characterized by a dense desmoplastic stroma with cancer-associated fibroblasts, pancreatic stellate cells, extracellular matrix, and immunosuppressive cells. Linked to this are genetic mutations in TP53 and immunosuppressive factors that act on T cells, resulting in a shortage of CD8+ T cells and limited expression of activation markers such as interferon-gamma. In this way, finding new strategies that make it possible to manipulate resistance mechanisms is necessary. Thus, techniques such as the use of TME modulators that block receptors and stromal molecules that generate resistance, the use of genetic manipulation in specific regions, such as microRNAs, the modulation of extrinsic and intrinsic factors associated with T cells, and, above all, therapeutic models that combine these modulation techniques constitute the promising future of PC therapy. Thus, this study aims to elucidate the main mechanisms of resistance to immunotherapy in PC and new ways of manipulating this process, resulting in a more efficient therapy for cancer patients and, consequently, a reduction in the lethality of this aggressive cancer.

Interrogating Matrix Stiffness and Metabolomics in Pancreatic Ductal Carcinoma Using an Openable Microfluidic Tumor-on-a-Chip.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense fibrotic stroma that contributes to aggressive tumor biology and therapeutic resistance. Current in vitro PDAC models lack sufficient optical and physical access for fibrous network visualization, in situ mechanical stiffness measurement, and metabolomic profiling. Here, we describe an openable multilayer microfluidic PDAC-on-a-chip platform that consists of pancreatic tumor cells (PTCs) and pancreatic stellate cells (PSCs) embedded in a 3D collagen matrix that mimics the stroma. Our system allows fibrous network visualization via reflected light confocal (RLC) microscopy, in situ mechanical stiffness testing using atomic force microscopy (AFM), and compartmentalized hydrogel extraction for PSC metabolomic profiling via mass spectrometry (MS) analysis. In comparing cocultures of gel-embedded PSCs and PTCs with PSC-only monocultures, RLC microscopy identified a significant decrease in pore size and corresponding increase in fiber density. In situ AFM indicated significant increases in stiffness, and hallmark characteristics of PSC activation were observed using fluorescence microscopy. PSCs in coculture also demonstrated localized fiber alignment and densification as well as increased collagen production. Finally, an untargeted MS study putatively identified metabolic contributions consistent with in vivo PDAC studies. Taken together, this platform can potentially advance our understanding of tumor-stromal interactions toward the discovery of novel therapies.

The nanobody targeting PD-L1 and CXCR4 counteracts pancreatic stellate cell-mediated tumour progression by disrupting tumour microenvironment

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal malignancy worldwide owing to its complex tumour microenvironment and dense physical barriers. Stromal-derived factor-1 (SDF-1), which is abundantly secreted by tumour stromal cells, plays a pivotal role in promoting PDAC growth and metastasis. In this study, we investigated the impact and molecular mechanisms of the anti-PD-L1&CXCR4 bispecific nanobody on the TME and their consequent interference with PDAC progression. We found that blocking the SDF-1/CXCR4 signalling pathway delayed the epithelial-mesenchymal transition in pancreatic cancer cells. Anti-PD-L1&CXCR4 bispecific nanobody effectively suppress the secretion of SDF-1 by pancreatic stellate cells and downregulate the expression of smooth muscle actin alpha(α-SMA), thereby preventing the activation of cancer-associated fibroblasts by downregulating the PI3K/AKT signaling pathway. This improves the pancreatic tumour microenvironment, favouring the infiltration of T cells into the tumour tissue. In conclusion, our results suggest that the anti-PD-L1&CXCR4 bispecific nanobody exerts an antitumor immune response by changing the pancreatic tumour microenvironment. Hence, the anti-PD-L1&CXCR4 bispecific nanobody is a potential candidate for pancreatic cancer treatment.

Abstract LB190: Cancer-stromal cells interaction dominates the expression of pro-tumor decreted protein chitinase-3-like-1 in pancreatic cancer

Abstract In the context of the tumor microenvironment (TME), a substantial increased secreted protein, chitinase-3 like-protein-1 (CHI3L1) has been observed, emanating from various cell types such as cancer cells, macrophages, and T cells. The development of an immunosuppressive and fibrotic TME has been implicated in CHI3L1-mediated cancer progression. Despite recognizing the potential for CHI3L1 blockade to facilitate cancer regression, there remains a limited understanding of the regulatory mechanisms governing the expression of this tumor-promoting protein within cancer cells. Our investigations, along with others, have identified low levels of CHI3L1 protein in normal epithelial cells, in stark contrast to its abundant presence in pancreatic cancer tumor specimens. We propose that the interaction between cancer and stromal cells in the TME induces CHI3L1 expression within cancer cells. To test this hypothesis, we established a co-culture system involving cancer and stromal cells including pancreatic stellate cells, macrophages and T cells to emulate the TME of pancreatic cancer. Subsequently, we observed a ten-fold increase in CHI3L1 expression in cancer cells upon exposure to co-culture media. Through secretomic analysis, C-X-C motif chemokine ligand 10 (CXCL10) emerged as a potential inducer of CHI3L1 expression in response to co-culture media. CXCL10/CXCR3 signaling has been reported to correlate with poor prognosis in pancreatic cancer, while their role in disease development is still unclear. Concurrently, Gene Set Enrichment Analysis (GSEA) identified the activation of TNFα signaling via NF-κB and TGF-β pathways in pancreatic cancer patients with high CHI3L1 expression. Notably, chromatin-immunoprecipitation and promoter activity assays validated the direct binding and transcriptional regulatory role of NF-κB, a downstream transcription factor of CXCL10, on CHI3L1 gene in cancer cells. In addition, epigenetic reprogramming occurred during co-culture media education. Our observations indicated demethylation of the CHI3L1 promoter during co-culture media exposure, a phenomenon confirmed to be mediated by STAT3 activation. In summary, this study elucidates, for the first time, the intricate crosstalk between cancer and stromal cells within the TME of pancreatic cancer as a pivotal factor for the transcriptional and epigenetic dysregulation of CHI3L1, which then contributes to TME remodeling and malignant pancreatic cancer progression. Citation Format: Pei-Chia (Peggy) Su, Yi-Ching Wang. Cancer-stromal cells interaction dominates the expression of pro-tumor decreted protein chitinase-3-like-1 in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB190.

Targeting ROCK2 improves macromolecular permeability in a 3D fibrotic pancreatic cancer microenvironment model

Pancreatic cancer is characterized by a densely fibrotic stroma. The fibrotic stroma hinders the intratumoral penetration of nanomedicine and diminishes therapeutic efficacy. Fibrosis is characterized by an abnormal organization of extracellular matrix (ECM) components, namely the abnormal deposition and/or orientation of collagen and fibronectin. Abnormal ECM organization is chiefly driven by pathological signaling in pancreatic stellate cells (PSCs), the main cell type involved in fibrogenesis. However, whether targeting signaling pathways involved in abnormal ECM organization improves the intratumoral penetration of nanomedicines is unknown. Here, we show that targeting transforming growth factor-β (TGFβ)/Rho-associated kinase (ROCK) 1/2 signaling in PSCs normalizes ECM organization and concomitantly improves macromolecular permeability of the fibrotic stroma. Using a 3-dimensional cell culture model of the fibrotic pancreatic cancer microenvironment, we found that pharmacological inhibition of TGFβ or ROCK1/2 improves the permeation of various macromolecules. By using an isoform-specific pharmacological inhibitor and siRNAs, we show that targeting ROCK2, but not ROCK1, alone is sufficient to normalize ECM organization and improve macromolecular permeability. Moreover, we found that ROCK2 inhibition/knockdown attenuates Yes-associated protein (YAP) nuclear localization in fibroblasts co-cultured with pancreatic cancer cells in 3D. Finally, pharmacological inhibition or siRNA-mediated knockdown of YAP normalized ECM organization and improved macromolecular permeability. Our results together suggest that the TGFβ/ROCK2/YAP signaling axis may be therapeutically targeted to normalize ECM organization and improve macromolecular permeability to augment therapeutic efficacy of nanomedicines in pancreatic cancer.

Abstract 2903: CTHRC1 induces PSCs (pancreatic stellate cells) into myCAFs (myofibroblast-like cancer-associated fibroblasts)

Abstract Collagen triple helix repeat containing-1 (CTHRC1) is a secreted protein that contributes to the progression of various cancers, including pancreatic cancer. The higher expression of CTHRC1 in tumor tissues is associated with poorer survival outcomes. However, its specific roles in tumor extracellular matrix (ECM) remodeling remain unclear. Our study aims to investigate the influences of CTHRC1 on pancreatic stellate cells (PSCs), a main source of ECM production in pancreatic cancer. The analyses of the publicly available pancreatic cancer patient data revealed that CTHRC1 is mainly expressed in cancer stroma and highly correlated with ECM-related genes. An in vitro study showed that more than 40% of these genes can be upregulated by CTHRC1. CTHRC1 specifically activated PSC into myofibroblast-like cancer-associated fibroblasts (myCAFs), which are characterized by a significantly upregulated POSTN gene expression. Periostin (coded by the POSTN gene) has a central role in the CTHRC1-PSCs-cancer metastasis axis. Furthermore, CTHRC1 promoted pancreatic cancer cell proliferation through PSC activation to a greater extent than via direct stimulation. Proof-of-concept experiments showed that the long-term (4-week) inhibition of CTHRC1 led to significant tumor suppression and ECM reduction, and also resulted in an unexpected shift in the CAF subtype from myCAFs to inflammatory CAFs (iCAFs). PSC activation was demonstrated to be the key molecular mechanism responsible for the tumor-promoting effects of CTHRC1, and CTHRC1 has a critical role in CAF subtype differentiation and tumor microenvironment (TME) remodeling. The inhibition of CTHRC1 as a therapeutic strategy for the treatment of pancreatic cancer warrants further investigation. Citation Format: Jin Gyeong Lee, Min Kyung Kang, Fen Jiang, Kyoungjin Ryu, Juwon Kim, Yun-Yong Park, Sang Seok Koh. CTHRC1 induces PSCs (pancreatic stellate cells) into myCAFs (myofibroblast-like cancer-associated fibroblasts) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2903.

Abstract 1584: The interplay between natural killer cells and pancreatic stellate cells in pancreatic ductal adenocarcinoma

Abstract Natural Killer (NK) cells have been shown to possess potent anti-tumor activity against multiple cancer types and consequently have been harnessed as a cellular cancer immunotherapy. Furthermore, targeting of pancreatic stellate cells has received increasing interest in the treatment of pancreatic ductal adenocarcinoma (PDAC), with several clinical trials testing the hypothesis that rendering activated pancreatic stellate cells/cancer associated fibroblasts (aPSC/CAF) into a quiescent state (qPSC) can enhance anticancer treatment. Despite this interest, our understanding of the interplay between these two cell types remains limited. We hypothesized that PSC/CAF may exhibit a dynamic interaction with NK cells that may be harnessed for therapeutic strategies and patient stratification. Employing a series of in vitro assays, we demonstrate a significant bi-directional interaction between natural killer cells and pancreatic stellate cells in the context of PDAC. NK cells were found to effectively target and eliminate both aPSC and qPSC, with PSC activation conferring some protection from NK-lysis. Furthermore, we highlight a significant myofibroblastic shift in PSCs regardless of previous activation status when cultured with NK cells; an interaction that was direct contact dependent. NK cells were also found to demonstrate cellular modulation of NKG2A and TIM3 in response to PSC co-culture. Drawing on Luminex ELISA analysis, we suggest that engagement of the IFN-γ signaling pathway may be driving phenotypic modulation. Global proteomic analyses demonstrated differential protein changes induced by NK cells in aPSC compared to qPSC, as well as alterations in the NK cell proteome upon such co-culture. Utilizing multiplex immunohistochemical analysis of PDAC patient samples, we highlight differential spatial distribution of phenotypically distinct NK cells and PSC/CAF subtypes in patient samples between long- (n=16) and short- (n=48) survivors. Moreover, NK-PSC proximity, and not total immune infiltrate, was found to play a prognostic role in PDAC. Our work provides a global overview of the cross-talk between NK and PSC in PDAC, demonstrating a significant bi-directional relationship which offers novel insights into potential therapeutic avenues. Furthermore, we demonstrate, for the first time to our knowledge, the prognostic role of NK-PSC proximity in patient survival and suggest its potential for patient stratification. Citation Format: Rachel Elizabeth Fincham, Parthiban Periasamy, Craig Ryan Joseph, Jia Meng, Jeffrey Chun Tatt Lim, Felicia Wee, Konstantinos Stasinos, Michelle R. Goulart, Jiangfeng Ye, Li Yen Chong, Denise Goh, Joe P. Yeong, Hemant M. Kocher. The interplay between natural killer cells and pancreatic stellate cells in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1584.

Abstract 2025: Zeb1 downregulation sensitizes pancreatic cancer-associated fibroblasts to killing by oncolytic reovirus through upregulation of the reovirus receptor junction adhesion molecule A

Abstract Pancreatic tumors display an abundance of cancer-associated fibroblasts (CAFs), which negatively affect prognosis and therapy response. Oncolytic virotherapy exploits viruses that preferentially lyse epithelial cancer cells as opposed to normal cells. Interestingly, we have observed that oncolytic reoviruses are able to infect and lyse CAFs, in addition to epithelial cancer cells. Targeting CAFs, in addition to cancer cells, could be advantageous to increase therapy effectiveness. It could serve as a conduit for viral spread and simultaneously disrupt the desmoplastic barrier around tumors, thereby also accelerating the influx of other therapeutics and immune cells. We previously found that the proneness of CAFs to lysis by oncolytic reovirus correlates with the cell surface expression levels of the reovirus entry receptor junction adhesion molecule A (JAM-A). However, most pancreatic CAFs do not express JAM-A. Therefore, a genome-wide CRISPR/Cas9 screen was employed to identify the genes regulating JAM-A expression on fibroblasts, which can subsequently be targeted to sensitize CAFs to reovirus. Pancreatic stellate cells with a moderate JAM-A expression level were transduced with a gRNA library making a knockout of one gene per cell. The highest and lowest JAM-A expressing cells were sorted and sequenced to identify the gRNAs that regulate JAM-A expression. Clonal CRISPR/Cas9-generated knockouts of a top negative regulator were generated and infected with reovirus, followed by cell viability assays to quantify their susceptibilities to reovirus-induced cell death. F11R, the gene encoding JAM-A, was identified as the top positive regulator of JAM-A expression in the CRISPR/Cas9 screen, verifying the validity of the screen. The top negative regulators identified were Fibroblast Growth Factor Receptor 1 (FGFR1) and Zinc finger E-box Binding homeobox 1 (Zeb1), thereby serving as potential therapeutic targets to sensitize CAFs to reovirus treatment. Using clonal Zeb1 knock-outs, Zeb1 was confirmed as a strong regulator of JAM-A expression. Zeb1 knockout in JAM-A negative pancreatic fibroblasts caused a robust upregulation of JAM-A and sensitized these inherently resistant fibroblasts to reovirus-directed cytolysis. Additionally, the clinically approved drug Mocetinostat, previously described to inhibit Zeb1, upregulated JAM-A expression on CAFs and increased cell lysis by reovirus. Altogether, our data show that Zeb1 is a strong negative regulator of JAM-A expression on fibroblasts and that Zeb1 inhibition can sensitize CAFs to reovirus-induced cell death. This research provides a rationale for combining Zeb1 inhibitory drugs with oncolytic reovirus treatment to improve killing of CAFs, which in turn could boost overall tumor eradication. Citation Format: Nicole Dam, Tom J. Harryvan, Bernhard Schmierer, Lukas J.A.C. Hawinkels, Vera Kemp. Zeb1 downregulation sensitizes pancreatic cancer-associated fibroblasts to killing by oncolytic reovirus through upregulation of the reovirus receptor junction adhesion molecule A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2025.

Abstract 489: Unlocking immune resistance and metastasis of PDAC via dual acting circularized nanodisc

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and deadliest forms of cancer. While major progress in immunotherapy has been made in recent years, it remains poorly effective in this malignancy. Two key players stand behind immune resistance of PDAC and inadequate efficacy of immunotherapy to suppress its metastasis. First, the immune-resistant pancreatic cancer stem cells (CSCs) evade immune surveillance acting as a source for tumor relapse and metastasis. This immune resistance stems from low immunogenicity of PDAC cells as well as the inaccessible CSCs that reside deep in the tumor core. Second, pancreatic cancer associated fibroblasts (CAFs) fuel metastasis of PDAC cells by suppressing their immune recognition and establishing pre-metastatic niche at distant organs to help their colonization. Our hypothesis is to overcome immune resistance of PDAC and inhibit early steps of its metastasis via simultaneous immunogenic eradication of immune resistant CSCs and switching immunosuppressive and pro-metastatic CAFs into quiescent ones. Therefore, we engineered ultrasmall circularized nanodiscs (cND) that encapsulate oncolytic peptide LTX-315 and fibroblast remodeling Notch signaling inhibitor. The engineered oncolytic LTX-315 loaded cND successfully induced immunogenic cell death (ICD) of both bulk PANC1 and pancreatic CSCs. Compared to non-treated and free LTX-315 treated cells, LTX-315 cND resulted in enhanced release of DAMPs including ATP and HMGB-1 as well as surface translocation of calreticulin. The ultrasmall size (9 nm) of cND enabled its deep tumor penetration to eradicate resistant CSCs, as revealed using 3D PDAC heterospheroids. In parallel, the Notch blocking cND promoted quiescence of the activated pancreatic stellate cells as denoted by downregulation of SMA, FAP and collagen. In KPC syngeneic PDAC mouse model, the dual oncolytic/Notch blocking cND combined with aPD-L1 therapy significantly suppressed tumor growth and prevented its metastasis to liver compared to vehicle-treated mice. Immunophenotyping study showed marked reduction in the abundance of CSCs and CAFs in the tumor treated with the cNDs. Moreover, the tumor infiltration of CD86+ MHCII+ dendritic cells and cytotoxic CD8+ T cells was enhanced after treatment with the cND. Overall, our dual strategy of targeting pancreatic CSCs and CAFs via oncolytic/Notch blocking cND boosted the anti-tumor and anti-metastatic efficacy of aPD-L1 therapy to eliminate primary PDAC tumor and suppress its metastasis. Citation Format: Ahmed Elzoghby, Hagar Emam, Seungbin Yim, Cuiyan Xin, Mahmoud Nasr. Unlocking immune resistance and metastasis of PDAC via dual acting circularized nanodisc [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 489.

Biodistribution and therapeutic efficacy of a gold nanoparticle-based targeted drug delivery system against pancreatic cancer

Pancreatic cancer is characterized by desmoplasia; crosstalk between pancreatic cancer cells (PCCs) and pancreatic stellate cells (PSCs) leads to the deposition of extracellular matrix proteins in the tumor environment resulting in poor vascularity. Targeting either PCCs or PSCs individually has produced mixed results, and there is currently no effective strategy to target both cell types simultaneously. Previously, we demonstrated, through in vitro cell culture experiments, that a specific gold nanoparticle-based nanoformulation containing the anti-EGFR antibody cetuximab (C225) as a targeting agent and gemcitabine as a chemotherapeutic agent effectively targets both PCCs and PSCs simultaneously. Herein, we extend our studies to test the ability of these in vitro tested nano formulations to inhibit tumor growth in an orthotopic co-implantation model of pancreatic cancer in vivo. Orthotopic tumors were established by co-implantation of equal numbers of PCCs and PSCs in the mouse pancreas. Among the various formulations tested, 5 nm gold nanoparticles coated with gemcitabine, cetuximab and poly-ethylene glycol (PEG) of molecular weight 1000 Da, which we named ACGP441000, demonstrated optimal efficacy in inhibiting tumor growth. The current study reveals an opportunity to target PCCs and PSCs simultaneously, by exploiting their overexpression of EGFR as a target, in order to inhibit pancreatic cancer growth.

Reactive Oxygen Species-Responsive Nanoparticles Toward Extracellular Matrix Normalization for Pancreatic Fibrosis Regression.

Pancreatic fibrosis (PF) is primarily characterized by aberrant production and degradation modes of extracellular matrix (ECM) components, resulting from the activation of pancreatic stellate cells (PSCs) and the pathological cross-linking of ECM mediated by lysyl oxidase (LOX) family members. The excessively deposited ECM increases matrix stiffness, and the over-accumulated reactive oxygen species (ROS) induces oxidative stress, which further stimulates the continuous activation of PSCs and advancing PF; challenging the strategy toward normalizing ECM homeostasis for the regression of PF. Herein, ROS-responsive and Vitamin A (VA) decorated micelles (named LR-SSVA) to reverse the imbalanced ECM homeostasis for ameliorating PF are designed and synthesized. Specifically, LR-SSVA selectively targets PSCs via VA, thereby effectively delivering siLOXL1 and resveratrol (RES) into the pancreas. The ROS-responsive released RES inhibits the overproduction of ECM by eliminating ROS and inactivating PSCs, meanwhile, the decreased expression of LOXL1 ameliorates the cross-linked collagen for easier degradation by collagenase which jointly normalizes ECM homeostasis and alleviates PF. This research shows that LR-SSVA is a safe and efficient ROS-response and PSC-targeted drug-delivery system for ECM normalization, which will propose an innovative and ideal platform for the reversal of PF.

COMP promotes pancreatic fibrosis by activating pancreatic stellate cells through CD36-ERK/AKT signaling pathways

BackgroundPancreatic fibrosis is one of the most important pathological features of chronic pancreatitis (CP) and pancreatic stellate cells (PSCs) are the key cells of fibrosis. As an extracellular matrix (ECM) glycoprotein, cartilage oligomeric matrix protein (COMP) is critical for collagen assembly and ECM stability and recent studies showed that COMP exert promoting fibrosis effect in the skin, lungs and liver. However, the role of COMP in activation of PSCs and pancreatic fibrosis remain unclear. We aimed to investigate the role and specific mechanisms of COMP in regulating the profibrotic phenotype of PSCs and pancreatic fibrosis. MethodsELISA method was used to determine serum COMP in patients with CP. Mice model of CP was established by repeated intraperitoneal injection of cerulein and pancreatic fibrosis was evaluated by Hematoxylin-Eosin staining (H&E) and Sirius red staining. Immunohistochemical staining was used to detect the expression changes of COMP and fibrosis marker such as α-SMA and Fibronectin in pancreatic tissue of mice. Cell Counting Kit-8, Wound Healing and Transwell assessed the proliferation and migration of human pancreatic stellate cells (HPSCs). Western blotting, qRT-PCR and immunofluorescence staining were performed to detect the expression of fibrosis marker, AKT and MAPK family proteins in HPSCs. RNA-seq omics analysis as well as small interfering RNA of COMP, recombinant human COMP (rCOMP), MEK inhibitors and PI3K inhibitors were used to study the effect and mechanism of COMP on activation of HPSCs. ResultsELISA showed that the expression of COMP significantly increased in the serum of CP patients. H&E and Sirius red staining analysis showed that there was a large amount of collagen deposition in the mice in the CP model group and high expression of COMP, α-SMA, Fibronectin and Vimentin were observed in fibrotic tissues. TGF-β1 stimulates the activation of HPSCs and increases the expression of COMP. Knockdown of COMP inhibited proliferation and migration of HPSCs. Further, RNA-seq omics analysis and validation experiments in vitro showed that rCOMP could significantly promote the proliferation and activation of HPSCs, which may be due to promoting the phosphorylation of ERK and AKT through membrane protein receptor CD36. rCOMP simultaneously increased the expression of α-SMA, Fibronectin and Collagen I in HPSCs. ConclusionIn conclusion, this study showed that COMP was up-regulated in CP fibrotic tissues and COMP induced the activation, proliferation and migration of PSCs through the CD36-ERK/AKT signaling pathway. COMP may be a potential therapeutic candidate for the treatment of CP. Interfering with the expression of COMP or the communication between COMP and CD36 on PSCs may be the next direction for therapeutic research.

Synthesis and biological activity of 11-Oxygenated and heterocyclic estrone analogs in pancreatic cancer monolayers and 3D spheroids

Pancreatic Ductal Adenocarcinoma (PDAC), representing over 90 % of pancreatic cancer diagnoses, is an aggressive disease with survivability among the worst of all cancers due to its difficulty in detection and its high metastatic properties. Current therapies for PDAC show limited success at extending life expectancies, primarily due to cancer resistance and lack of patient-specific targeted therapies. This work highlights the design and evaluation of estrone-derived analogs with both heterocyclic side-chain functionality and 11-oxygenated functionality for use in pancreatic cancer. First-round heterocyclic analogs show preliminary promise in AsPC-1 and Panc-1 cell lines, with IC50 values as low as 10.16 ± 0.83 µM. Their success, coupled with design choices from other studies, led to the synthesis of novel 11-hydroxyl and 11-keto estrone analogs that show potent in-vitro toxicity against various pancreatic cancer models. The three most cytotoxic analogs, KA1, KA2, and KA9 demonstrated low micromolar activities in both MTT and CellTiter assays in three pancreatic cancer cell lines: AsPC-1, Panc-1, and BxPC-3, as well as in a co-culture of Panc-1 and pancreatic stellate cells. IC50 values for KA9 (4.17 ± 0.90, 5.28 ± 1.87, and 5.70 ± 0.65 µM respectively) shows consistency in all cell lines tested. KA9 is also able to cause an increase in caspases 3 and 7 activity, key markers for apoptosis, at non-cytotoxic concentrations. Additional work was performed by generating 3D pancreatic cancer spheroids to better modulate the pancreatic tumor microenvironment, and KA9 continued to show the best IC50 values (21.0 and 24.3 µM) in both cell types tested. KA9 was also able to prevent the growth of spheroids whereas the standard chemotherapy, Gemcitabine, could not, suggesting that it may be a potent analog for future development of treatments. Molecular dynamic simulations were also performed to confirm biological findings and uncovered that KA9′s preferential binding location is in the active site pocket of key proteins involved in cytotoxicity.

Pancreatic Cancer Treatment Targeting the HGF/c-MET Pathway: The MEK Inhibitor Trametinib.

Pancreatic cancer is characterized by fibrosis/desmoplasia in the tumor microenvironment, which is primarily mediated by pancreatic stellate cells and cancer-associated fibroblasts. HGF/c-MET signaling, which is instrumental in embryonic development and wound healing, is also implicated for its mitogenic and motogenic properties. In pancreatic cancer, this pathway, along with its downstream signaling pathways, is associated with disease progression, prognosis, metastasis, chemoresistance, and other tumor-related factors. Other features of the microenvironment in pancreatic cancer with the HGF/c-MET pathway include hypoxia, angiogenesis, metastasis, and the urokinase plasminogen activator positive feed-forward loop. All these attributes critically influence the initiation, progression, and metastasis of pancreatic cancer. Therefore, targeting the HGF/c-MET signaling pathway appears promising for the development of innovative drugs for pancreatic cancer treatment. One of the primary downstream effects of c-MET activation is the MAPK/ERK (Ras, Ras/Raf/MEK/ERK) signaling cascade, and MEK (Mitogen-activated protein kinase kinase) inhibitors have demonstrated therapeutic value in RAS-mutant melanoma and lung cancer. Trametinib is a selective MEK1 and MEK2 inhibitor, and it has evolved as a pivotal therapeutic agent targeting the MAPK/ERK pathway in various malignancies, including BRAF-mutated melanoma, non-small cell lung cancer and thyroid cancer. The drug's effectiveness increases when combined with agents like BRAF inhibitors. However, resistance remains a challenge, necessitating ongoing research to counteract the resistance mechanisms. This review offers an in-depth exploration of the HGF/c-MET signaling pathway, trametinib's mechanism, clinical applications, combination strategies, and future directions in the context of pancreatic cancer.

ITGB6 promotes pancreatic fibrosis and aggravates the malignant process of pancreatic cancer via JAK2/STAT3 signaling pathway.

Integrin β6 (ITGB6) is upregulated in multiple tumor types and elevated ITGB6 levels have been detected in patients with chronic pancreatitis. However, the role of ITGB6 in pancreatic fibrosis and cancer remains to be elucidated. In the present study, ITGB6 expression was assessed using western blotting and qRT-PCR. Besides, cell proliferation, cycling, migration, and invasion were evaluated using CCK-8, flow cytometry, wound healing, and transwell assays, respectively. The expression of fibrosis and JAK2/STAT3 signaling markers was detected by western blotting and immunofluorescence analysis. Moreover, nude mice were subcutaneously injected with co-cultured cell suspensions to establish an in vivo model. The results showed that ITGB6 was highly expressed in pancreatic cancer tissues and TGF-β-induced pancreatic stellate cells (PSCs). Inhibition of ITGB6 expression in PSCs resulted in clear inhibition of activated PSC proliferation, migration, and fibrogenesis. Additionally, reduced ITGB6 expression inhibits the JAK2/STAT3 signaling pathway. Interestingly, activators of the JAK2/STAT3 signaling pathway reversed the effects of ITGB6 disruption on PSCs. Activated PSCs notably promoted the proliferation, invasion, and migration of pancreatic cancer cells in a co-culture assay. In contrast, activated PSCs with low ITGB6 expression failed to significantly affect the malignancy of pancreatic cancer cells. Moreover, in vivo results showed that interference with ITGB6 inhibited the activation of PSCs and promoted the development of pancreatic cancer. Silencing ITGB6 inhibited the proliferation, migration, and fibrosis-like effects of activated PSCs and indirectly inhibited the metastasis and malignant process of pancreatic cancer by inhibiting the JAK2/STAT3 signaling pathway. Therefore, ITGB6 is a potential candidate target for pancreatic cancer prevention and treatment.

Disruption of Super-Enhancers in Activated Pancreatic Stellate Cells Facilitates Chemotherapy and Immunotherapy in Pancreatic Cancer.

One major obstacle in the drug treatment of pancreatic ductal adenocarcinoma (PDAC) is its highly fibrotic tumor microenvironment, which is replete with activated pancreatic stellate cells (a-PSCs). These a-PSCs generate abundant extracellular matrix and secrete various cytokines to form biophysical and biochemical barriers, impeding drug access to tumor tissues. Therefore, it is imperative to develop a strategy for reversing PSC activation and thereby removing the barriers to facilitate PDAC drug treatment. Herein, by integrating chromatin immunoprecipitation (ChIP)-seq, Assays for Transposase-Accessible Chromatin (ATAC)-seq, and RNA-seq techniques, this work reveals that super-enhancers (SEs) promote the expression of various genes involved in PSC activation. Disruption of SE-associated transcription with JQ1 reverses the activated phenotype of a-PSCs and decreases stromal fibrosis in both orthotopic and patient-derived xenograft (PDX) models. More importantly, disruption of SEs by JQ1 treatments promotes vascularization, facilitates drug delivery, and alters the immune landscape in PDAC, thereby improving the efficacies of both chemotherapy (with gemcitabine) and immunotherapy (with IL-12). In summary, this study not only elucidates the contribution of SEs of a-PSCs in shaping the PDAC tumor microenvironment but also highlights that targeting SEs in a-PSCs may become a gate-opening strategy that benefits PDAC drug therapy by removing stromal barriers.

Chronic exposure to BDE-47 aggravates acute pancreatitis and chronic pancreatitis by promoting acinar cell apoptosis and inflammation.

The effect of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a persistent environmental pollutant commonly used as a flame retardant in various consumer products, on pancreatitis has not been clearly elucidated, although it has been reported to be toxic to the liver, nervous system, and reproductive system. Acute pancreatitis (AP) and chronic pancreatitis (CP) models were induced in this study by intraperitoneal injection of caerulein. The aim was to investigate the impact of BDE-47 on pancreatitis by exposing the animals to acute (1 week) or chronic (8 weeks) doses of BDE-47 (30 mg/kg in the low-concentration group and 100 mg/kg in the high-concentration group). Additionally, BDE-47 was utilized to stimulate mouse bone marrow-derived macrophages, pancreatic primary stellate cells, and acinar cells in order to investigate the impact of BDE-47 on pancreatitis. In vivo experiments conducted on mice revealed that chronic exposure to BDE-47, rather than acute exposure, exacerbated the histopathological damage of AP and CP, leading to elevated fibrosis in pancreatic tissue and increased infiltration of inflammatory cells in the pancreas. In vitro experiments showed that BDE-47 can promote the expression of the inflammatory cytokines Tnf-α and Il-6 in M1 macrophages, as well as promote acinar cell apoptosis through the activation of the PERK and JNK pathways via endoplasmic reticulum stress. The findings of this study imply chronic exposure to BDE-47 may exacerbate the progression of both AP and CP by inducing acinar cell apoptosis and dysregulating inflammatory responses.

Nicotinamide adenine dinucleotide phosphate oxidase in pancreatic diseases: Mechanisms and future perspectives.

Pancreatitis and pancreatic cancer (PC) stand as the most worrisome ailments affecting the pancreas. Researchers have dedicated efforts to unraveling the mechanisms underlying these diseases, yet their true nature continues to elude their grasp. Within this realm, oxidative stress is often believed to play a causal and contributory role in the development of pancreatitis and PC. Excessive accumulation of reactive oxygen species (ROS) can cause oxidative stress, and the key enzyme responsible for inducing ROS production in cells is nicotinamide adenine dinucleotide phosphate hydrogen oxides (NOX). NOX contribute to pancreatic fibrosis and inflammation by generating ROS that injure acinar cells, activate pancreatic stellate cells, and mediate macrophage polarization. Excessive ROS production occurs during malignant transformation and pancreatic carcinogenesis, creating an oxidative microenvironment that can cause abnormal apoptosis, epithelial to mesenchymal transition and genomic instability. Therefore, understanding the role of NOX in pancreatic diseases contributes to a more in-depth exploration of the exact pathogenesis of these diseases. In this review, we aim to summarize the potential roles of NOX and its mechanism in pancreatic disorders, aiming to provide novel insights into understanding the mechanisms underlying these diseases.

Modulating pancreatic cancer microenvironment: The efficacy of Huachansu in mouse models via TGF-β/Smad pathway

Ethnopharmacological relevanceHuachansu (HCS) is a traditional Chinese medicine obtained from the dried skin glands of Bufo gargarizans and clinical uses of HCS have been approved in China to treat malignant tumors. The traditional Chinese medicine theory states that HCS relieves patients with cancer by promoting blood circulation to remove blood stasis. Clinical observation found that local injection of HCS given to pancreatic cancer patients can significantly inhibit tumor progression and assist in enhancing the efficacy of chemotherapy. However, the material basis and underlying mechanism have not yet been elucidated. Aim of the studyTo investigate the therapeutic potential of HCS for the treatment of pancreatic cancer in in situ transplanted tumor nude mouse model. Furthermore, this study sought to elucidate the molecular mechanisms underlying its efficacy and assess the impact of HCS on the microenvironment of pancreatic cancer. To identify the antitumor effect of HCS in in situ transplanted tumor nude mouse model and determine the Chemopreventive mechanism of HCS on tumor microenvironment (TME). MethodsUsing the orthotopic transplantation nude mouse model with fluorescently labeled pancreatic cancer cell lines SW1990 and pancreatic stellate cells (PSCs), we examined the effect of HCS on the pancreatic ductal adenocarcinoma (PDAC) microenvironment based on the transforming growth factor β (TGF-β)/Smad pathway. The expression of TGF-β, smad2, smad3, smad4, collagen type-1 genes and proteins in nude mouse model were detected by qRT-PCR and Western blot. ResultsHCS significantly reduced tumor growth rate, increased the survival rate, and ameliorated the histopathological changes in the pancreas. It was found that HCS concentration-dependently reduced the expression of TGF-β1 and collagen type-1 genes and proteins, decreased the expression of Smad2 and Smad3 genes, and downregulated the phosphorylation level of Smad2/3. Additionally, the gene and protein expression of Smad4 were promoted by HCS. Further, the promoting effect gradually enhanced with the rise of HCS concentration. ConclusionsThe results demonstrated HCS could regulate the activity of the TGF-β/Smad pathway in PDAC, improved the microenvironment of PDAC and delayed tumor progression. This study not only indicated that the protective mechanism of HCS on PDAC might be attributed partly to the inhibition of cytokine production and the TGF-β/Smad pathway, but also provided evidence for HCS as a potential medicine for PDAC treatment.